Fig. 2.

(A) Finding k_intact. The rate constant of hydrogen peroxide diffusion across the membrane was determined by adding hydrogen peroxide to a suspension of HeLa cells and measuring the extracellular concentration of peroxide using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) absorbance based assay. (B) Finding k_catalase. HeLa cells were permeabilized with increasing concentrations of digitonin and lysates were incubated with 10  mM H₂O₂. Depletion of peroxide was measured at 240  nm and the first order rate constant was determined. The plateau before the rise in kinetic activity indicates conditions where the peroxisome is not disrupted. (C) Finding k_GPx. HeLa cells lysed with 0.1% Triton were incubated with 1.3–3  mM of GSH, 1.1  U/mL of glutathione reductase, 1  mM NaN₃, 0.3  mM NADPH and 35  µM hydrogen peroxide. NADPH depletion, as an indicator of hydrogen peroxide depletion, was measured at 340  nm. Hydrogen peroxide depletion kinetics between 2 and 15  µM were then fitted to an integrated rate equation and k_GPx was determined.